Purified linear polypeptide epitopes from walnuts and associated methods

ABSTRACT

A purified Ig-E binding immunogenic polypeptide the amino acid sequence of which consists of SEQ ID NO:1 is disclosed. The purified amino acid sequence is a major allergen of the walnut  Juglans regia . Also described are a diagnostic test kit and use of the purified polypeptide in tests for identifying patients having an allergy to walnuts. Mutations of the disclosed polypeptide, as well as nucleic acid sequences encoding therefor are claimed as part of the invention.

RELATED APPLICATION

This application claims priority from and is a division of applicationSer. No. 11/829,120 filed on Jul. 27, 2007, now U.S. Pat. No. 7,763,721,which was a division of Ser. No. 10/251,918, filed on Sep. 20, 2002, nowU.S. Pat. No. 7,250,489, and which claimed priority from provisionalapplication Ser. No. 60/324,161, filed on Sep. 20, 2001, all of whichare incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to the field of allergies to nuts and,more particularly, to a purified linear epitope polypeptide from walnutsand associated methods.

SEQUENCE LISTING

A Sequence Listing is submitted herewith as an ASCII compliant text filethroughout the Electronic Filing System; the name of the text file is“Purified Linear Polypeptide Epitope from Walnuts”: the file was createdon Aug. 4, 2006 and is 3 kilobytes in size: this Sequence Listing isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

It is estimated that up to 8% of children less than 3 years of age and2% of adults are affected by food allergies (reviewed in ¹). Whilefood-induced allergic reactions are the most common cause of outpatientanaphylaxis,² the majority of severe reactions of this kind are causedby peanuts and tree nuts. ^(3, 4) Most plant food allergens can be foundamong pathogenesis-related proteins, seed storage albumins andglobulins, and a-amylase and protease inhibitors.

A previous study by Teuber et al. ⁶ documented the cloning andsequencing of a gene encoding Jug r 1, a major allergen in the Englishwalnut, Juglans regia. Jug r 1 is a 2S albumin seed storage protein andpossesses important homologies in amino acid sequence with other 2Salbumin proteins from Brazil nut, cottonseed, castor bean, and mustard.6Like many 2S albumins, Jug r 1 is synthesized as a precursor protein andcleaved into a large and small subunit joined by disulfide bridges. Itis currently believed that patients with life-threatening allergies towalnuts and other tree nuts will rarely become tolerant of these foods ⁷and consequently face a lifetime of avoidance. ⁸ Avoidance, however, isoften difficult due to the ubiquitous nature of these foods and thepotential for cross-contamination during their processing. ⁷

Many approaches to allergen immunotherapy under investigation today arebased on a detailed knowledge of the amino acids found in IgE-reactiveB-cell epitopes. ^(9, 10, 11, 12) These reactive sites can be eitherlinear or conformational. Typically, a linear epitope contains a stretchof contiguous amino acids spanning 5-10 residues in the antigen, whileresidues distant in the primary sequence but proximate in the foldedprotein comprise a conformational epitope. ¹³ In recent years, the genesencoding several proteins with allergenicity, such as Jug r 1, have beencloned and expressed using molecular biology techniques.^(6, 14, 15, 16) Despite the rapidly increasing number of recombinantallergens, relatively few IgE-reactive B-cell epitopes have beendefined. ¹⁷⁻²⁹

REFERENCES CITED

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A synthetic    hexadecapeptide derived from allergen M imposing allergenic and    antigenic reactivity. Scand J Immunol 1980; 12:171-5.-   18. Atassi H, Atassi M Z. Antibody recognition of ragweed allergen    Ra3: localization of the full profile of the continuous antigenic    sites by synthetic overlapping peptides representing the entire    protein chain. Eur J Immunol 1986; 16:229-35.-   19. Mazur G, Baur X, Modrow S, Becker W M. A common epitope on major    allergens from non-biting midges (Chironomidae). Mol Immunol 1988;    25:1005-10.-   20. van't H of W, van Milligen F J, van den Berg M, Lombardero M,    Chapman M D, Aalberse R C. Epitope mapping of the cat (Felis    domesticus) major allergen Fel d I by overlapping synthetic peptides    and monoclonal antibodies against native and denatured Fel d I.    Allergy. 1993; 48:255-63.-   21. Helm R M, Cockrell G, Connaughton C, West C M, Herman E, Sampson    H A, Bannon G A, Burks A W. 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SUMMARY OF THE INVENTION

With the foregoing in mind, the present invention advantageouslyprovides a purified Ig-E binding immunogenic polypeptide comprising awalnut IgE-binding epitope. Peanut and tree nut allergies can be lifethreatening, and appear to be growing in prevalence. Jug r 1, a 2Salbumin seed storage protein, was previously characterized as a majorEnglish walnut food allergen. Accordingly, we sought to identify thelinear immunoglobulin E (IgE)-binding epitopes of Jug r 1 and determinewhich, if any, amino acids are necessary for this binding to occur. Theaim of the research leading to the present invention was to screen thelarge and small subunits of the English walnut allergen Jug r 1, insearch for linear IgE-binding epitopes. We have discovered a singleimmunodominant IgE-binding epitope, residing in the large subunit, andhave identified the core amino acids necessary for this binding tooccur.

Pools of sera from walnut-allergic patients and overlapping peptidessynthesized on an activated cellulose membrane were used to screen forIgE-binding epitopes. Mutational analysis of the immunodominant epitopewas carried out via single and multi-site amino acid substitutions.Inhibition assays were performed using affinity-purified IgE, solubleforms of the epitope peptide, and the recombinant 2S albumin, rJug r 1.

One immunodominant-linear epitope having twelve amino acid residues wasidentified, and is set forth herein as SEQ ID NO:1. Amino-acid mutationsto the epitope demonstrated that the residues RGEE, at positions 4 to 7,were required for IgE binding. Probing of this epitope with each of 20patients' sera revealed 15 were positive. Binding of patients' IgE tothe epitope was inhibited with a soluble form of the peptide, howeversoluble peptide did not completely inhibit the binding of IgE to theintact rJug r 1.

One major linear IgE-reactive epitope and its required core amino acidresidues have been identified. Mutation of any of these core amino acidsresulted in loss of IgE binding to the epitope and therefore suggeststhe feasibility of reducing allergenicity in genetically modifiedwalnuts by engineering nuts with a modified epitope sequence. Althoughthis approach may be feasible, nevertheless, strong evidence for theexistence of conformational epitopes was also obtained, thus, a fullynon-allergenic bioengineered nut may require additional modifications.

BRIEF DESCRIPTION OF THE DRAWINGS

Some of the features, advantages, and benefits of the present inventionhaving been stated, others will become apparent as the descriptionproceeds when taken in conjunction with the accompanying drawings inwhich:

FIG. 1 shows the entire 64-amino acid length of the large subunit (FIG.1A; SEQ ID NO:4) and 26-amino acid length of the small subunit (FIG. 1B;SEQ ID NO:5);

FIG. 2 dot-blot assay showing IgE from 20 individual walnut-sensitivepatients binding to the 20-kDa His-tagged rJug r 1;

FIG. 3 shows results of a study of the entire lengths of both the largeand small subunits of Jug r 1 carried out by probing overlapping solidphase synthetic peptides with sera from 20 patients randomly assigned tofour pools; each pool recognized three adjacent peptides from the largesubunit; two peptides were recognized very strongly (#'s 11 and 12) andone less so (#10); no peptides were identified from the small subunit(FIG. 3A); a common sequence, GLRGEEM, was observed in all three largesubunit peptides (FIG. 3B; SEQ ID NO:3);

FIG. 4 results of the 20 patients tested, five exhibiting strongrecognition of the epitope peptide, six moderate, four weak, and fiveshowing no recognition; none reacted with the (−) peptide;

FIG. 5 shows binding of IgE to the positive (E1) peptide inunfractionated patient serum, progressively less binding in the firsttwo eluate fractions, and no binding in the third, indicating completeremoval of the E1-reactive Ab by the column (FIG. 5A); E1-adsorbed serum(effluent) showing no reactivity to the E1 peptide, confirms removal ofE1-specific IgE from the serum; eluate and effluent fractions were thenused in dot-blot assays to determine if IgE Abs to the E1 epitoperepresent the bulk of the rJug r 1-reactive IgE, with results showingbinding of IgE to rJug r 1, from total sera and the first two eluate(anti-E1) fractions, but not the third (anti-E1-depleted) (FIG. 5B);

FIG. 6 is a table showing results of mutational analysis of the Jug r 1IgE binding epitope designated E1, also referred to as SEQ ID NO:1; and

FIG. 7 displays in tabular form the amino acid sequence for the presentimmunogenic walnut polypeptide (SEQ ID NO:1) and its correspondingcodons.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention will now be described more fully hereinafter withreference to the accompanying drawings, in which preferred embodimentsof the invention are shown. Unless otherwise defined, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionpertains. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, suitable methods and materials are described below.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including any definitions,will control. In addition, the materials, methods and examples given areillustrative in nature only and not intended to be limiting. Amino acidsare referred to by their standard single or three letter abbreviation.Similarly, nucleic acids are identified by their single letter code, asknown in the art.

Accordingly, this invention may, however, be embodied in many differentforms and should not be construed as limited to the illustratedembodiments set forth herein. Rather, these illustrated embodiments areprovided so that this disclosure will be thorough and complete, and willfully convey the scope of the invention to those skilled in the art.Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

LIST OF ACRONYMS AND ABBREVIATIONS USED

ECL: enhanced chemiluminescence;

IgE: immunoglobulin E;

NC: nitrocellulose;

RT: room temperature;

ddH20: distilled-deionized water;

BSB: buffered saline borate;

TBS: tris-buffered saline;

TBS-T: tris-buffered saline with 0.2% Tween-20;

PBS: phosphate buffered saline;

GST: glutathione-S-transferase;

DMF: N,N-dimethylformamide;

Fmoc: 9-fluorenlymethoxy carbonyl-derived;

SDS-PAGE: sodium dodecylsulfate-polyacrylamide gel electrophoresis; and

Overnight: o/n.

Methods

Human Sera

The study was approved by the Human Subjects Review Committee at theUniversity of California, Davis. Sera from 20 walnut-allergic patientswith a convincing history of life-threatening systemic allergicreactions to walnuts, positive ImmunoCAP assays (Pharmacia, Inc.,Diagnostics, Columbus, Ohio), and evidence of IgE against rJug r 1 (seebelow) were used in this study. Control sera were obtained from atopicpatients with no history of walnut sensitivity, and one patient withlife-threatening reactions to walnuts, positive ImmunoCAP assay,positive serum IgE immunoblot to walnut proteins, but negative forbinding to the 2S albumin.

Expression and Isolation of rJug r 1

Two protocols for isolation of rJug r 1 were used. In the first, used toproduce rJug r 1 for screening patient sera by Western blotting, the Jugr 1 insert was subcloned into the pPROEXHT expression vector (Gibco BRL,Invitrogen Life Technologies, Carlsbad, Calif.), and transformed into E.coli BL21-Codon Plus (Stratagene Inc., La Jolla, Calif.). Cells weregrown overnight (o/n) at 37° C. in 5 ml 2×YT broth with 50 μg/mlampicillin and chloramphenicol. This was then added to 500 ml 2×YT brothand grown to an A₆₀₀ of 1.0. Isopropyl thiogalactose was added to 0.5mM, and the culture was grown at 37° C. for 4 hrs. Cells were pelleted,washed with 0.02 M phosphate buffered saline (PBS; pH 7.3), andresuspended in BRL Lysis Buffer (Gibco BRL). Lysozyme was added to 500μg/ml on ice for 15 min, followed by the addition of sarkosyl to 1% w/v.The cells were sonicated and the insoluble matter pelleted bycentrifugation at 12,000 g for 20 min at 4° C. Ni-NTA resin (Gibco BRL)was added in BRL Buffer A (Gibco BRL) to the supernatant and allowed toincubate o/n at 4° C. The resin was washed 4× with BRL Buffer A and theHis-tagged fusion protein then eluted with BRL Buffer C (Gibco BRL)containing 0.25% sarkosyl. In the second protocol, rJug r 1 wasexpressed in fusion with glutathione-S-transferase (GST) and purifiedfollowing the Frangioni and Neel protocol with slight modifications aspreviously described by Teuber et al ^(6, 30). This GST-rJug r 1 fusionprotein was used in all dot-blot assays, as described below.

Immunoblotting of rJug r 1

rJug r 1 samples were either subjected to sodiumdodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) andelectrophoretically transferred to nitrocellulose (NC), or blotteddirectly onto the membrane. For SDS-PAGE analysis of rJug r 1, sampleswere boiled for five min in sample buffer (60 mM Tris-HCl, pH 6.8, 2%SDS, 10% (v/v) glycerol, 0.01% (w/v) bromophenol blue) andelectrophoresis was carried out at 8 mA constant current using a SE600Vertical Slab Gel Unit (Pharmacia Biotech, Piscataway, N.J.). AnSDS-PAGE gel, 13% monomer acrylamide concentration, with 1 μg protein/4mm was used for immunoblotting as previously described.⁶ Protein wastransferred to a 0.22 mm NC membrane (MSI, Westborough, Mass.) o/n at30V using a TE 42 Transphor Electro-Transfer Unit (Pharmacia Biotech,Piscataway, N.J.). The blot was cut into 4 mm wide strips and blockedfor 1 hr at room temperature (RT) in PBS/3% nonfat dry milk/0.2% TritonX-100 (TX-100). Diluted sera, 1:5 v:v in the blocking buffer, were addedto the strips and incubated o/n at RT. The strips were then washed for20 min 3× in PBS/0.01% TX-100 and incubated o/n at RT with equinepolyclonal ¹²⁵I-anti-human IgE (Hycor Biomedical Inc., Garden Grove,Calif.) diluted 1:5 in the nonfat milk buffer. The strips were washedfor 20 min 3× and exposed to Kodak Biomax x-ray film (Kodak, Rochester,N.Y.) at −70° C. for 48 hours. All incubations were carried out withrocking.

For dot-blot assays, a graphite pencil was used to circumscribe 4 mm×8mm elipses on a dry 0.4 μm NC membrane (Shleicher & Schuell, Keen,N.H.). The NC was incubated in distilled-deionized water (ddH₂O) on arocking table for 5 min and allowed to air dry before each dot wasloaded with 0.11 μg of rJug r 1 in 2 μl of ddH₂O. The antigen-loaded NCwas then rinsed in ddH₂O as described above, placed protein side up on 3MM paper (Whatman Intl. Ltd., Maidstone, England), and dried under a60-watt light until all moisture was removed. Once dry, dots were rinsedfor 2 min in Tris-buffered saline (TBS: 20 mM Tris, 137 mM NaCl, pH 7.6)containing 0.2% Tween-20, blocked for 1 hr at RT in TBS-T containing 2%bovine-serum albumin (BSA, Sigma, St. Louis, Mo.), and then incubatedo/n at 4° C. with sera diluted 1:40 in TBS-T. Membranes were then washedonce for 15 min, and 3× for 5 min in TBS-T prior to being incubated for1 hr at RT with horseradish peroxidase-labeled goat-anti-human IgE(Biosource International, Camarillo, Calif.) diluted 1:2000 in TBS-T.Washing in TBS-T was repeated as above and the reactive dots wereidentified after a 5-min incubation in Enhanced Chemiluminescence Plus(ECL+, Amersham Pharmacia, Piscataway, N.J.), prepared followingmanufacturer's instructions, and subsequent exposure to Kodak X-OMATx-ray film.

Solid-Phase Peptide (SPOTs) Synthesis

Based on the published amino acid sequence of Jug r 1 and ourunpublished data determining the subunit cleavage sites,⁶ twenty-five13-amino acid peptides, offset by three amino acids, were synthesizedwhich corresponded to the entire 64-amino acid length of the largesubunit (FIG. 1A) and 26-amino acid length of the small subunit (FIG.1B). Additional peptides bearing the target epitope (QGLRGEEMEEMV), SEQID NO:1, mutated forms of this peptide, and a non-IgE binding peptide(LSQRGLQSSSV), SEQ ID NO:6, were also synthesized. Thirteen versions ofthe target peptide were created via a single site alanine substitutionat each position along the amino acid sequence, and six mutated peptideswere synthesized using multiple alanine substitutions, as shown in FIG.6.

Peptides were synthesized on derivatized cellulose sheets using9-fluorenlymethoxy carbonyl-derived (Fmoc) amino acids as described bythe manufacturer (Genosys Biotechnologies, Inc., The Woodlands, Tex.).Briefly, cellulose membranes containing free hydroxy groups wereesterfied with an Fmoc-amino acid dissolved in 1-methyl-2 pyrrolidione.The coupling reaction was followed by washing in N,N-dimethylformamide(DMF) and the amino acids were acetylated with acetic anhydride. Themembranes were deprotected by washing in DMF and incubation with asolution of 20% piperidine in DMF followed by another wash in DMF.Coupling, acetylation, and deprotection steps were repeated for eachcycle. During the final cycle, a mixture of dichloromethane,trifluoroacetic acid, and triisobutylsilane (1:1:0.05) was used todeprotect the acid-labile amino acid side chains.

IgE Binding to Solid Phase Synthetic Peptides (SPOTs Analysis)

The peptide-containing membranes were washed in TBS and incubated o/n atRT in blocking solution as directed by the manufacturer (GenosysBiotechnologies, Inc., The Woodlands, Tex.). Membranes were then washedin TBS-T for 10 min and incubated o/n at 4° C. with individual patient'sserum or pooled patients' sera diluted 1:5 in blocking buffer (totalsera:blocking buffer). This incubation was followed by three 5-minwashes in TBS-T and an o/n (4° C.) incubation with ¹²⁵I-anti-human IgE(Hycor Biomedical Inc., Garden Grove, Calif.) diluted 1:10 in a mixtureof PBS, 5% nonfat dry milk, and 0.05% Tween-20. Three final 10 minwashes in PBS/0.05% Tween-20 were performed and IgE-peptide reactivityidentified after a 48 hr exposure at −70° C. to Kodak Biomax x-ray film.

Soluble Epitope Peptide Synthesis

A soluble form of an identified IgE-reactive peptide (QGLRGEEMEEMV; SEQID NO: 1) was synthesized by Fmoc protocols on an automated peptidesynthesizer (Model 433A, Applied Biosystems, Foster City, Calif.).

Isolation and Inhibition of Epitope-Specific IgE

The epitope-reactive IgE was isolated from patients' sera by affinitychromatography. A 0.5 ml pool of patients' sera, known to be reactive tothe reactive synthetic solid-phase epitope peptide, was slowly added toa 5 ml disposable polypropylene column (Pierce Chemical Company,Rockford, Ill.) containing cyanogen-bromide-activated beads (Sigma, St.Louis, Mo.) (to which 5 mg of the reactive peptide had been covalentlycoupled, as described by the manufacturer) and the effluent collected.Bound IgE was eluted with the addition of 0.2 M glycine sulfate, pH 2.3,collected in a beaker containing 10 μl of 1% BSA in buffered salineborate (BSB), and subsequently neutralized with 1.0 M Tris. The columnwas then rinsed with BSB and the initial effluent re-passed over thecolumn. The process was repeated for a total of 3×, yielding threeeluates and the column effluent.

To test the specificity and reactivity of the epitope-specific IgE,control, pooled and fractionated patients' sera were pre-incubated o/nat 4° C. with different amounts (70, 7.0, and 0.7 μg) of the solubleepitope peptide, or a soluble non-specific peptide. The pre-incubatedsera were used to probe either solid phase IgE-reactive andnon-IgE-reactive peptides or rJug r 1. Detection of IgE binding to thesolid-phase synthetic peptides and dot-blotted rJug r 1 was detectedusing ¹²⁵I-anti-human IgE and ECL+, respectively, as described earlier.

IgE Binding to rJug r 1

IgE from 20 individual walnut-sensitive patients was shown to bind the20-kDa His-tagged rJug r 1 (FIG. 2). Three showed only faint binding,while 17 sera gave moderate or strong signals. Recombinant Jug r 1 waspreviously shown to completely inhibit binding to the native 2S albuminand substantially inhibit binding of patient sera IgE to English walnutin walnut ImmunoCAP assays (up to 84% of measurable anti-walnut IgE),⁶thus the folding appears to preserve most of the presumed conformationalepitopes present on the native mature protein.

Identification and Recognition of IgE-Reactive Linear Epitopes on rJug r1

The entire lengths of both the large and small subunits of Jug r 1 werestudied by probing overlapping solid phase synthetic peptides with serafrom 20 patients randomly assigned to four pools. Each pool recognizedthree adjacent peptides from the large subunit; two peptides wererecognized very strongly (#s 11 and 12) and one less so (#10); nopeptides were identified from the small subunit (FIG. 3A). A commonsequence, GLRGEEM (SEQ ID NO:7), was observed in all three large subunitpeptides (FIG. 3B). A fourth partially overlapping sequence (#9, FIG.3A) showed a slight positive reaction. Some other peptides showed slightpositive signals in some assays (i.e. #7, FIG. 3A) but were notreproducible, leading us to examine only the identified dominant linearepitope-bearing peptides. Additional peptides were tested in whichalanine was substituted at each of the 12 amino residues of theIgE-reactive peptide, QGLRGEEMEEMV (SEQ ID NO: 1). In addition, peptideswere tested with varying numbers of alanines substituted at the N- andC-termini. Together, these data demonstrated that the core amino acidsRGEE, at positions 36 to 39, and an additional glutamic acid residue atposition 42, were necessary for maximum IgE binding to occur (FIG. 6).SPOTs containing a 12-amino acid peptide (QGLRGEEMEEMV; SEQ ID NO: 1),bearing the immunodominant epitope, designated E1, and SPOTs containinga negative control (−) peptide (LSQRSQQQCRQ; SEQ ID NO:8), selected fromthe large subunit of Jug r 1 were used to test the degree of individualreactivity and specificity of allergic and control sera.

Of the 20 patients tested, five exhibited strong recognition of theepitope peptide, six moderate, four weak, and five showed norecognition; none reacted with the (−) peptide (FIG. 4).

Isolation and Inhibition of E1-Specific IgE

To determine if the identified epitope, designated E1, is the majorepitope recognized by patients' sera, we separated E1-specific IgE fromthe total antiserum and tested both fractions for reactivity withrJugr 1. Epitope-specific IgE was isolated from patient serum by passageover an E1-affinity column. To assure removal of anti-E1 Abs, the threesequential eluate and the serum effluent fractions were first assayedagainst our solid-phase IgE binding (+) and non-IgE binding (−)peptides. FIG. 5A shows binding of IgE to the positive (E1) peptide inunfractionated patient serum, progressively less binding in the firsttwo eluate fractions, and no binding in the third, indicating completeremoval of the E1-reactive Ab by the column. Significantly, theE1-adsorbed serum (effluent) also showed no reactivity to the E1peptide, confirming removal of E1-specific IgE from the serum. Theeluate and effluent fractions were then used in dot-blot assays todetermine if IgE Abs to the E1 epitope represent the bulk of the rJug r1-reactive IgE. As expected, binding of IgE to rJug r 1, from total seraand the first two eluate (anti-E1) fractions, but not the third(anti-E1-depleted), was demonstrated (FIG. 5B). However, there wasconsiderable rJug r 1-specific IgE remaining in the effluent (anti-E1depleted fraction), demonstrating the presence of additional IgE Abspecific for one or more conformational epitopes.

To further examine the specificity of both our peptide-reactivefractions and unfractionated Jug r 1-reactive IgE, a soluble form of theepitope peptide was pre-incubated with whole patient serum as well asthe E1-specific IgE Abs in an attempt to inhibit their reaction withboth our solid-phase peptides and rJugr 1. Varying amounts (70, 7.0, 0.7μg) of the peptide were incubated with whole patients' sera prior toprobing the E1 epitope (positive) and negative solid-phase peptides.Partial to complete inhibition was achieved (FIG. 5C), demonstratingthat the solid and fluid phase versions of the peptide are similarlyrecognized. The amount of soluble peptide (70 μg) needed to completelyinhibit binding of E1-specific IgE to the solid phase version of thisepitope was similarly used for inhibition studies involvingunfractionated patient serum IgE and affinity column purified IgEfractions against the rJug r 1. Inhibition was again observed in theeluate (E1 epitope-specific) IgE fractions.

These data demonstrate that the epitope is similarly recognized in boththe peptide and the recombinant protein. However, IgE reactivity in bothwhole patient serum and the E1-adsorbed effluent fraction reacted withrJug r 1 (FIG. 5D), again demonstrating the existence of additional(presumably conformational) IgE-reactive epitopes on rJug r 1.

DISCUSSION

On a global basis walnuts rank second in tree nut production.³¹ Inchildren, anaphylactic reactions to peanut and tree nuts are responsiblefor most fatal and near-fatal food allergic reactions.^(3, 32) In arecent study, 32 fatal cases of anaphylactic reactions to foods reportedto a national registry were analyzed and it was determined that up to31% were due to tree nuts.³³ The severity and prevalence of some foodinduced allergic reactions has prompted investigation to identify andcharacterize offending allergens.

Jug r 1, a major allergen in the English walnut, has previously beencharacterized as a 2S albumin seed storage protein.⁶ 2S albumins arefound in almost all edible seeds. In view of the importance of thisclass of protein as food allergens and the possibility ofcross-reactivity among class-related proteins,³⁴ a detailed knowledge ofthese proteins would be valuable. In particular, given the importantrole allergen-specific IgE plays in the allergic reaction, determinationof allergen-specific IgE binding epitopes, be they linear orconformational, appears to be of great importance for gaining a betterunderstanding of the allergenic nature of foods and for possibletherapeutic intervention.

We have found a single linear IgE-reactive epitope and have defined itscore amino acid residues. To date, no common structural character oflinear-IgE epitopes has been identified (reviewed in³⁵), but this couldchange as more epitope mapping studies are completed. A key step in theallergic reaction is the binding of at least two IgE Abs to amultivalent allergen. The fact that we have found only one linearepitope on Jug r 1 is unique in that all previously analyzed allergenshave contained multiple linear IgE-binding sites (reviewed in³⁵).Although we cannot rule out the existence of weakly binding peptidesthat are undetected by our methods, the affinity of such peptides wouldbe much lower as judged by the high intensity of the signal on theepitope E1. Our findings, in combination with the knowledge thatallergens must be multivalent to elicit an allergic response, pointtowards the existence of conformational epitopes on Jug r 1 as well.Although it is qualitative data, it is noteworthy that all 20 patients'sera bound rJug r 1 (in FIG. 2), whereas only 15 bound the linearepitope, with many of these exhibiting disproportionately faint binding.This too, is evidence that conformational epitopes are relevant and keyin IgE binding for some patient sera. In fact, it has been estimatedthat most protein epitopes are conformational.³⁶ It is worthwhile tonote that the labeling of an epitope as “linear”, based on the use ofsynthetic epitopes, is imprecise because the identified epitope may be afragmented part of a larger discontinuous epitope (reviewed in³⁵).

Clinically, insight into the IgE-binding epitopes of allergens, linearor conformational, is extremely useful. Identifying the immunodominantlinear IgE-binding epitope of Jug r 1 may lead to better designs forwalnut-allergy therapy. Currently there is no generally agreed upontreatment for IgE-mediated food allergy and, therefore, completeavoidance of the food is recommended.³⁷ However, with tree nuts, such aswalnuts, avoidance is often difficult since equipment used for foodprocessing is often shared and the implicated allergen may, therefore,be present in trace amounts or may be added to foods where its presencewas not expected by the consumer. Because tree nut allergies are rarelyresolved, reactions can be severe, and accidental ingestion is almostinevitable, life for afflicted patients can be fraught with anxiety(reviewed in³⁸). Some new approaches to allergen immunotherapy anddiagnosis include: the use of blocking peptides to inhibit thetriggering of IgE-mediated hypersensitivity reactions, immunization withspecific peptides to alter the type of immune response elicited,allergen reengineering to enhance protective responses, and the use ofdefined peptides in diagnostic assays. ^(9, 10, 11, 12) A common factorfor the application of these approaches is a required knowledge of theamino acids in the IgE-reactive sites of the allergen. We have mappedthe major linear IgE epitope of Jug r 1 and identified the principalcore amino acid residues within the epitope. Additional studies areplanned to identify the conformational epitopes on this allergen.

Accordingly, the invention disclosed herein includes a purified Ig-Ebinding immunogenic polypeptide of walnuts, the amino acid sequence ofwhich consists of SEQ ID NO:1. The immunogenic polypeptide of theinvention may also include at least one mutation in said amino acidsequence, wherein said at least one mutation is selected from adeletion, a substitution, and an addition. The polypeptide may alsocomprise at least one mutation in said amino acid sequence which reducesthe polypeptide's IgE-binding capacity while maintaining at leastminimal immunogenicity. The skilled will recognize that the inventivepolypeptide may also comprise at least two SEQ ID NO:1.

The invention additionally includes a test for detecting a walnutallergy in a patient, said test comprising contacting the patient withan effective amount of a purified polypeptide consisting of SEQ ID NO:1for detecting the allergy. In one embodiment of the test contactingcomprises injecting the purified polypeptide below the skin of thepatient. For use in this test, the purified polypeptide may comprise atleast one mutation in said sequence, and the mutation is selected from adeletion, a substitution, and an addition.

A further embodiment of the invention includes a diagnostic test fordetecting anti-walnut IgE in a patient to thereby indicate an allergy towalnuts. The diagnostic comprises reacting patient's serum with apurified polypeptide the amino acid sequence of which consists of atleast one SEQ ID NO:1; separating the polypeptide from unreacted patientserum; reacting the polypeptide with a labeled human IgE-reactive agentafter separating from unreacted patient serum; separating thepolypeptide from unreacted labeled human IgE-reactive agent; anddirectly or indirectly detecting the labeled human IgE-reactive agentbound to the polypeptide after separating from unreacted agent tothereby indicate presence in the patient's serum of anti-walnut IgE. Foruse in this diagnostic test, as also noted previously, the amino acidsequence may further comprise at least one mutation selected from adeletion, a substitution, and an addition.

Yet another embodiment of the invention includes a test kit forscreening patients for allergy to walnuts. The kit comprises a firstreagent containing at least one purified polypeptide whose amino acidsequence consists of SEQ ID NO:1. The kit also contains a second reagentcontaining at least one labeled human IgE-reactive agent, for example,anti human IgE, or others known or to be developed. In the test kit, thefirst reagent preferably comprises a solid phase carrying the purifiedpolypeptide.

Those skilled in the art will readily recognize that from a known aminoacid sequence such as SEQ ID NO:1, we may readily prepare an isolatednucleic acid sequence and degenerate variants thereof which encode apolypeptide consisting of SEQ ID NO:1, as shown in FIG. 7. Such anucleic acid may further comprise a sequence which encodes at least onemutation of SEQ ID NO:1, wherein said mutation is selected from adeletion, a substitution, and an addition. The nucleic acid may be anisolated cDNA and degenerate variants thereof which encode a polypeptideconsisting of SEQ ID NO:1, or at least one mutation thereof wherein saidmutation is selected from a deletion, a substitution, and an addition.

In the drawings and specification, there have been disclosed a typicalpreferred embodiment of the invention, and although specific terms areemployed, the terms are used in a descriptive sense only and not forpurposes of limitation. The invention has been described in considerabledetail with specific reference to these illustrated embodiments. It willbe apparent, however, that various modifications and changes can be madewithin the spirit and scope of the invention as described in theforegoing specification and as defined in the appended claims.

1. A test for detecting a walnut allergy in a patient, the testcomprising: contacting the patient with an effective amount of apurified polypeptide consisting of SEQ ID NO: 1; and detecting whethersaid contacting the patient results in an allergic response, wherein anallergic response is indicative of a walnut allergy in the patient. 2.The test of claim 1, wherein contacting comprises contacting the skin ofthe patient.
 3. The test of claim 1, wherein contacting comprisesinjecting the patient.